The overall objective of the proposed research is to determine the nature of trans (cell-to cell) and cis (same cell surface) interactions of protocadherin (Pcdh) cell surface proteins. The majority of mammalian Pcdhs are present in three large gene clusters, and the organization of these clusters leads to the generation of enormous single cell diversity. This diversity is thought to function as a molecular barcode for individual neurons, which allows cells to distinguish between self and non-self. In particular, the clustered Pcdhs have been shown to be required for dendritic self-avoidance and other aspects of neural circuit assembly in the mouse brain and spinal cord. This function is likely to require homophilic interactions between distinct Pcdh isoforms at the surface of opposing plasma membranes. Initially, a cell-cell aggregation assay will be used to carry out a comprehensive analysis of Pcdh isoform homophilic specificity. A selected subset of Pcdh Alpha, Beta and guama isoforms tagged with fluorescent labels will be cloned and transfected into mammalian cells in culture. Cells transfected with the same or different Pcdh isoforms will be mixed and cell aggregation quantitated by the size of the immunofluorescent cell aggregates by fluorescent microscopy. The trans-homophilic interaction domains will be identified by constructing and testing Pcdh isoforms missing specific extracellular domains. Once identified the interacting ectodomains will be subjected to biophysical analyses to determine the multimeric state and homophilic binding affinities. Characterization of cis Pcdh interactions will be accomplished through the transfection of cells with multiple Pcdh isoforms, followed by cell-cell aggregation assays. Domain deletion studies will be carried out to map the cis- interacting regions and these regions subjected to biophysical analyses. Once identified, trans dimeric homophilic domains will be expressed at high levels, and the dimeric complexes crystallized and subjected to three-dimensional atomic structure analyses. Structural hypothesis based on the three dimensional structure will be tested using site-directed mutagenesis and cell aggregation assays as a means a correlating structure and function. Finally, attempts will be made to produce and crystallize full-length Pcdhs and sub regions that encompass the cis interface and their structures determined by x-ray crystallography. If successful, the proposed studies will provide deep mechanistic insights into the role of the clustered Pcdhs in mediating self-avoidance and other aspects of neural circuit assembly. In addition, the studies may reveal the existence and nature of a Pcdh-mediated molecular recognition code.